Stimulation of Fibroblast Proliferation by Insoluble Gadolinium Salts

The purpose of this study was to assess insoluble salts containing gadolinium (Gd³⁺) for effects on human dermal fibroblasts. Responses to insoluble Gd³⁺ salts were compared to responses seen with Gd³⁺ solubilized with organic chelators, as in the Gd³⁺-based contrast agents (GBCAs) used for magnetic resonance imaging. Insoluble particles of either Gd³⁺ phosphate or Gd³⁺ carbonate rapidly attached to the fibroblast cell surface and stimulated proliferation. Growth was observed at Gd³⁺ concentrations between 12.5 and 125 μM, with toxicity at higher concentrations. Such a narrow window did not characterize GBCA stimulation. Proliferation induced by insoluble Gd³⁺ salts was inhibited in the presence of antagonists of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways (similar to chelated Gd³⁺) but was not blocked by an antibody to the platelet-derived growth factor receptor (different from chelated Gd³⁺). Finally, high concentrations of the insoluble Gd³⁺ salts failed to prevent fibroblast lysis under low-Ca²⁺ conditions, while similar concentrations of chelated Gd³⁺ were effective. In conclusion, while insoluble Gd³⁺ salts are capable of stimulating fibroblast proliferation, one should be cautious in assuming that GBCA dechelation must occur in vivo to produce the profibrotic changes seen in association with GBCA exposure in the subset of renal failure patients that develop nephrogenic systemic fibrosis.     

Microalgae engineering toolbox: Selectable and screenable markers

Engineering microalgae has opened a new era for plant biologists and biotechnologists. Microalgae had been proved as a promising candidate for the production of biopharmaceuticals, nutraceuticals, antioxidants, antimicrobial and antiviral compounds, in dyeing and food industry as well for biofuel production. Genetic transformation of some important microalgae has been successful, but several other potential microalgae species still need scientific attention. The success of the genetic transformation depends mainly on the utilization of the selectable and screenable markers. Like for other higher crop plants, several useful markers have been reported for microalgae transformation. In this follow-up, we compared different marker genes for genetic engineering of approximately all the industrially important microalgae. We have discussed the expression host, the targeted genome, appropriate selection agent, as well as the transformation method. Genetic transformation is an expensive and labor intensive process and this review will aid to shorten the time span by providing a database of appropriate markers for microalgae research which could serve as a guide for those involved in the genetic engineering of microalgae.     

Cyanobacteria in rice soils

Cyanobacteria were recovered from each of 38 soil samples collected from local rice fields. Of the 84 species belonging to 31 genera that were isolated, 42 were heterocystous diazotrophic species belonging to 14 genera and the remaining were non-heterocystous. Fischerella, Nostoc and Calothrix were widespread.Z.U.M. Khan is with the Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh. Z.N. Tahmida Begum and M.Z. Hossain are with the Department of Botany and R. Mandal is with the Department of Soil Science, bot at the University of Dhaka, Dhaka-1000, Bangladesh;     

INTERCELLULAR ADHESIONS IN THE POLLEN-STIGMA SYSTEM: POLLEN CAPTURE,GRAIN BINDING,AND TUBE ATTACHMENTS

Four independent pollen-stigma binding forces are differentiated after pollen contacts stigmas of Brassica oleracea var. capitata. Identification is based on their different rates and sequence of appearance during gametophyte development; on their differential occurrence after compatible and incompatible pollinations; and on their different stabilities to NaOH and hexane. The first binding force develops most rapidly, begins seconds after pollen-stigma contact, is complete within minutes, occurs on compatible or incompatible papillae, and dissociates in methanol. A second slower binding reaction begins about 15 min after contact, continues for at least 90 min (when pollen tubes emerge), results in a binding structure termed the ocreatine, develops only on compatible papillae, and is dissociated by NaOH. A third attractive mechanism binds the tip of the emerging tube to a cuticle, is detected only on incompatible papillae, and is not dissociated by NaOH or methanol. Ocreatine formation and tube development beyond the emergence stage are prevented by the incompatibility response. A fourth attraction mechanism occurs between the surfaces of papilla and elongating tubes. Reviews of physical and biochemical evidence indicate that van der Waals forces and enzymatically mediated lipid polymerization are alternatives to agglutination as mechanisms for binding male gametophyte to papilla.     

Enzyme immunoassay (EIA) in the rapid diagnosis of gonorrhoea

The diagnostic value of a new, modified enzyme immunoassay (EIA) (Gonozyme; Abbott Laboratories, North Chicago III) was evaluated for the rapid antigenic detection of Neisseria gonorrhoeae in endocervical and urethral specimens. EIA results were compared with those of Gram stain (GS) and conventional culture tests. EIA sensitivity and specificity for male patients attending dermatovenerological clinic were 100% and 96.8% respectively in comparison to 86.7% and 96.8% obtained by Gram staining. For female Obstetrics-Gynaecology patients EIA sensitivity of 100% was highly significant compared to 50% sensitivity by the Gram stain. In culture, 30 strains of N. gonorrhoeae were isolated from 125 male specimens and 2 from 105 specimens from females; this suggests a prevalence of N. gonorrhoeae of 24% in males and 1.9% in females. In vitro antibiotic sensitivity testing indicated 55% resistance to penicillin and 43% to ampicillin in these isolated strains; all were sensitive to erythromycin/tetracycline. 12% of the strains were beta-lactamase producers.     

Enhanced degradation of phenol in floating treatment wetlands by plant-bacterial synergism

Phenol is a commonly found organic pollutant in industrial wastewaters. Its ecotoxicological significance is well known and, therefore, the compound is often required to be removed prior to discharge. In this study, plant-bacterial synergism was established in floating treatment wetlands (FTWs) in an attempt to maximize the removal of phenol from contaminated water. A common wetland plant, Typha domingensis, was vegetated on a floating mat and augmented with three phenol-degrading bacterial strains, Acinetobacter lwofii ACRH76, Bacillus cereus LORH97, and Pseudomonas sp. LCRH90, to develop FTWs for the remediation of water contaminated with phenol. All of the strains are known to have phenol-reducing properties, and grow well in FTWs. Results showed that T. domingensis was able to remove a small amount of phenol from the contaminated water; however, bacterial augmentation enhanced the removal potential significantly, i.e., 0.146 g/m²/day vs. 0.166 g/m²/day, respectively. Plant biomass also increased in the presence of bacterial consortia; and inoculated bacteria displayed successful colonization/survival in the rhizosphere, root interior and shoot interior of the plant. Similarly, highest reduction in chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), and total organic carbon (TOC) was achieved by the combined application of plants and bacteria. The study demonstrates that the plant-bacterial synergism in a FTW may be a more effective approach for the remediation of phenol-contaminated water.     

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon?+?and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~?25.5% and ~?6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.     

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Background

Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.

Methods

In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.

Results

We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.

Conclusions

Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.